Use of bucillamine in the treatment of gout

ABSTRACT

Disclosed are pharmaceutical compositions comprising, bucillamine, including bucillamine and allopurinol or colchicine, or pharmaceutically acceptable salts or solvates thereof, together with one or more pharmaceutically acceptable carriers, diluents and excipients. Methods for use of the said compositions in the treatment of gout and metabolic syndrome are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage entry under 35 U.S.C. §371 orInternational Application No. PCT/CA2013/050882, filed Nov. 19, 2013,which claims the benefit of priority of Australian Application No.2012905072, filed Nov. 20, 2012.

TECHNICAL FIELD

The present invention relates to pharmaceutical compositions comprisingbucillamine and their use for the treatment of gout.

BACKGROUND OF THE INVENTION

It is estimated that 8.3 million people suffer from active gout in theU.S.¹ The incidence and prevalence of gout is rising. This is due tofactors such as an increase in the aged population, many of whom takethiazide diuretics and prophylactic aspirin that promote hyperuricaemiaand lifestyle factors characterized by diets that include excessivefructose and alcohol intake, physical inactivity and abdominal fataccumulation which favor hyperuricaemia.^(2,3)

The impaired renal excretion of uric acid is the dominant cause ofhyperuricaemia in the majority of patients with gout.⁴ The existence ofgenetic variants within a transport gene, SLC2A9, have been reportedthat explain 1.7% to 5.3% of the variance in serum urate concentrationsin a Croatian population sample.⁵ SLC2A9 variants have also beenassociated with a low fractional excretion of urate and the presence ofgout in several other European population samples.⁶ The SLC2A9 gene isfound on human chromosome 4 and encodes the facilitative glucosetransporter 9 (Glut9), which is a unique hexose and high-capacity uratetransporter.⁷ It has been shown that Glut9 is expressed in thebasolateral membrane of hepatocytes and in both apical and basolateralmembranes of the distal nephron in the mouse.⁸ Glut9 sustains uratereabsorption in the kidney independently of the other known uratetransporters URAT1, OAT1, and OAT3.⁸

It has been found recently that human ATP-binding cassette, subfamily G,member 2 (ABCG2), encoded by the ABCG2 gene, is located in the brushborder membrane of kidney proximal tubule cells, where it mediates renalurate secretion.⁹ Introduction of a mutation encoded by a common ABCG2SNP (rs2231142) in Xenopus oocytes resulted in a 53% reduction in uratetransport rates compared with wild-type ABCG2. The data obtained in apopulation-based study of 14,783 individuals support rs2231142 as thecausal variant in the region, exhibiting highly significant associationswith urate concentration. These findings suggest that this SNP has asignificant pathogenic role in at least 10% of all gout cases in whitepersons.⁹ It has been confirmed in a study in Japanese patients thatcommon nonfunctional mutations of ABCG2 are involved in the pathology ofgout.¹⁰

The risk of developing gout is related to the degree ofhyperuricaemia^(11, 12) and increases rapidly after the serum urateconcentration exceeds ˜400 μmol/L which is close to the level at whichmonosodium urate (MSU) crystals precipitate out of serum in vitro.¹³ Ithas been established that phagocytosed intracellular MSU crystals aredetected in the cytoplasm by the NALP3 inflammasome in monocytes ormacrophages.¹⁴ The result is activation of caspase-1, which initiatesIL-1β maturation and secretion. In turn, interleukin IL-1β secretionproduces various pro-inflammatory mediators, which elicit neutrophilinflux into the joints.¹⁴ The results of in-vivo studies have confirmedthat IL-1β and its pathway is crucially associated with the inflammatoryresponse induced by MSU crystals, suggesting that IL-1β is a pivotalmediator of inflammation in acute gout as well as chronic gout and a keytherapeutic target.^(15, 16.)

The first-line urate-lowering therapy for the treatment of gout over thepast four decades has been allopurinol which lowers serum urate levelsby the inhibition of xanthine oxidase.¹⁷ The FDA guidelines recommendincreasing the dose progressively from an initial dose of 100 mg a dayto a maximum of 800 mg a day until the target serum urate (SU) level of≦6 mg/dL ((˜357 μmol/L) is achieved.¹⁸ However, the vast majority ofallopurinol prescriptions are for doses of 300 mg a day or less. Thissituation has been promoted by the persistence of longstandingnon-evidence based dosing guidelines that were originally designed toavoid the allopurinol hypersensitivity syndrome without anyconsideration for efficacy.¹⁸

Febuxostat (Ulloric®, Takeda) is a urate lowering therapy recentlyapproved by the FDA for the treatment of chronic gout. It is anon-purine-analogue inhibitor of xanthine oxidase that lowerscirculating uric acid levels.¹⁷ It was found that febuxostat was moreeffective than allopurinol in attaining appropriate SU levels (<360μmol/L), but 59% of patients who previously failed to normalise uricacid levels <360 μmol/L) on allopurinol also failed on febuxostat 80mg/day.¹⁹ Febuxostat is an alternative for patients for whom allopurinolis relatively contraindicated, due to a lack of evidence-based studies,particularly in mild to moderate renal dysfunction where dosemodification is not required. The main side effects of febuxostatinclude raised hepatic enzymes and a small increase in the rate ofserious cardiovascular events which preclude its use in ischaemic orcongestive heart failure.

The ageing of the population and the proliferation of unhealthylifestyles together with the sub-optimal use of allopurinol anduncertainty concerning the place of febuxostat emphasises the need fornew drugs for the treatment of gout.

The present inventor has realised that the recent advances in themolecular genetics of renal urate transporters together with thediscovery of the inflammasome may offer the opportunity to develop noveltherapeutic agents based on the identification of specific targets thatare involved in the pathology of gout.

Bucillamine, (Rimatil®, N-(2-mercapto-2-methylpropionyl)-L-cysteine) ismanufactured by Santen Pharmaceutical Co. Ltd. It is a disease-modifyinganti-rheumatic drug which is used as a first-line treatment forrheumatoid arthritis in Japan.²⁰ Bucillamine is a member of a group oflow molecular weight, cysteine-derived thiol donors that includesN-acetylcysteine and N-2-mercaptopropionyl glycine.^(21, 22) Thesecompounds readily enter cells through the cysteine transport pathway andexert their antioxidant effect by maintaining the endogenousglutaredoxin (Gtx) and thioredoxin (TRx) systems in a reduced state bytransfer of thiol groups.^(21, 22) Bucillamine contains two donatablethiol groups, making it a considerably more potent antioxidant thanN-acetylcysteine or N-2-mercaptopropionyl glycine which each containonly one thiol group.²³⁻²⁵ The present inventor has appreciated that inaddition to its direct antioxidant action, bucillamine also increasesthe transcriptional activity of Nrf2.²⁶

The physiological importance of ABCG2 in humans is illustrated by thelarge differences in SU levels and the prevalence of gout caused bygenetic variation in ABCG2. It is therefore, a potential target for newuricosuric agents in the treatment of gout.^(9, 10) It has been shownthat ABCG2 (BCRP) is induced by the Nrf2 activators oltipraz in primaryhuman hepatocytes²⁷ and tert-butylhydroquinone in HepG2 cells.²⁸

The multidrug resistance protein 4 (MRP4/ABCC4) has been identified as aunidirectional efflux pump for urate with multiple allosteric substratebinding sites located at the apical membrane of kidney proximaltubules.²⁹ The treatment of wild-type and Nrf2-null mice with oltiprazand butylated hydroxyanisole demonstrated that the induction of ABCC4was Nrf2-dependent.³⁰ Oltipraz also induces ABCC4 (MRP4) mRNA andprotein expression in HepG2 cells and primary human hepatocytes via theNrf2 transcription pathway.³¹

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is not to betaken as an admission that any or all of these matters form part of theprior art base or were common general knowledge in the field relevant tothe present invention as it existed before the priority date of eachclaim of this application.

SUMMARY OF THE INVENTION

The present inventor hypothesized that bucillamine could have similareffects as oltipraz on ABCC4.

The Nrf2-dependent induction of renal MRP4 (ABCC4) protein has observedin cisplatin-treated wild-type mice.³² The present inventor hypothesizedthat renal ABCC4 may be unregulated by Nrf2 activating compounds such asolitipraz or bucillamine.

The xanthine oxidase inhibitor allopurinol and its active metaboliteoxypurinol have both been shown to stimulate MRP4 (ABCC4) mediated uratetransport suggesting a new mechanism that may contribute to their uratelowering effect.³³ The present inventor hypothesized that the use of acombination of allopurinol and an Nrf2 activator such as bucillamine hasa synergistic effect in lowering the SU.

It has been reported previously that fenofibrate has a rapidurate-lowering effect in patients with hyperuricaemia and gout beingtreated with allopurino1.³⁴ Fenofibrate increases renal uric acidclearance but the exact mechanism has not been determined.^(34, 35) Onepossibility is that it may induce urate transporters through theactivation of Nrf2.³⁶ Moreover, a number of studies have reported thatcoffee consumption (greater than 5 cups of coffee) is associated withlower concentrations of urate^(36, 37) and a decreased risk of gout.³⁸There is evidence that coffee activates Nrf2^(39, 40) which suggest thatit may increase urate excretion through the activation of renal uratetransporters.

The prevalence of the metabolic syndrome is high in patients withgout.^(41, 42) It has been found that uric acid excretion is lower ingout patients with the metabolic syndrome.⁴³ Moreover, this disturbanceappears to be related to the severity of the metabolic syndrome.⁴³ Theactivation of Nrf2 by oltipraz decreases insulin resistance and obesitywhich are both elements of the metabolic syndrome. ⁴⁴ In another study,the oleanolic triterpenoid CDDO-im, which is a particularly potent Nrf2activator, prevented the development of obesity.⁴⁵ The present inventorhas realised that bucillamine and other activators of Nrf2 may increaseurate excretion through the attenuation of the metabolic syndrome.

It has been shown recently that uric acid triggers the association ofNALP3 with thioredoxin-interacting protein (TxNIP) in a reactive oxygenradical-dependent manner.⁴⁶ In unstimulated cells, TxNIP isconstitutively bound to and inhibited by thioredoxin (Trx). Following anincrease in oxidative stress, this complex dissociates and TxNIP bindsto NALP3 promoting the assembly and oligomerisation of the inflammasome.In support of such an activation mechanism, the knockdown of thioredoxinpotentiates inflammasome activation.⁴⁶

In work on the present invention, the inventor has appreciated thatBucillamine may attenuate the activation of NALP3 by MSU crystals.Without being bound to any particular theory, it could act through thedirect transfer of thiol groups to Trx.^(21, 22) In addition bucillaminecan activate Nrf2²⁶ and the binding of the transcription factor Nrf2 tothe ARE of the Trx gene is an induction mechanism for thioredoxin.⁴⁷

The FDA recently approved colchicine (Colcrys™, URL Pharma) for theprophylaxis of acute gout flares.⁴⁸ Colchicine has been used for manyyears as an unapproved drug with no FDA-approved prescribinginformation, dosage recommendations, or drug interaction warnings.Colchicine targets the initial stage of inflammation in gout⁴⁹ but actsupstream of the inflammasome.⁵⁰ The inventor has realized thatbucillamine and colchicine has a synergistic effect in attenuatinginflammation during acute gout flares.

In work on the present invention, the inventor surprisingly found forthe first time, that bucillamine has a potent uricosuric effect.Particularly when used in conjunction with allopurinol, bucillamineprovides a promising combination for the treatment of gout. Moreover,bucillamine has potent anti-inflammatory effects that may beparticularly useful in the management of acute gout flares. In addition,the inventor found that bucillamine together with colchicine produced asynergistic effect, which may offer a new combination therapy for goutwith increased efficacy and fewer side effects than with colchicinealone.

Accordingly, in a first aspect of the invention there is provided, apharmaceutical composition comprising bucillamine or a pharmaceuticallyacceptable salt or solvate thereof, together with one or morepharmaceutically acceptable carriers, diluents and excipients.

Accordingly, in a second aspect of the invention there is provided, apharmaceutical composition comprising bucillamine or a pharmaceuticallyacceptable salt or solvate thereof and allopurinol or a pharmaceuticallyacceptable salt or solvate thereof, together with one or morepharmaceutically acceptable carriers, diluents and excipients.

According to a third aspect of the invention there is provided, apharmaceutical composition comprising bucillamine or a pharmaceuticallyacceptable salt or solvate thereof and colchicine or a pharmaceuticallyacceptable salt or solvate thereof, together with one or morepharmaceutically acceptable carriers, diluents and excipients.

According to a fourth aspect of the invention there is provided, amethod for the treatment of gout in a mammal comprising administering atherapeutically effective amount of bucillamine or a pharmaceuticallyacceptable salt or solvate thereof and allopurinol or a pharmaceuticallyacceptable salt or solvate thereof, to a mammal in need thereof.

According to a fifth aspect of the invention there is provided, a methodfor the treatment of gout in a mammal comprising administeringtherapeutically effective amount of bucillamine or a pharmaceuticallyacceptable salt or solvate thereof and colchicine or a pharmaceuticallyacceptable salt or solvate thereof, to a mammal in need thereof.

According to a sixth aspect of the invention there is provided, a use ofbucillamine or a pharmaceutically acceptable salt or solvate thereof andallopurinol or a pharmaceutically acceptable salt or solvate thereof, inthe manufacture of a medicament for the treatment of gout.

According to a seventh aspect of the invention there is provided, a useof bucillamine or a pharmaceutically acceptable salt or solvate thereofand colchicine or a pharmaceutically acceptable salt or solvate thereof,in the manufacture of a medicament for the treatment of gout.

According to a eighth aspect of the invention there is provided, apharmaceutical composition according to the first or second aspects ofthe invention for use in the treatment of gout.

The present invention demonstrates, for the first time, that bucillaminehas a uricosuric effect in hyperuricaemic mice that may be attributableto the enhancement of uric acid excretion. Moreover, the combination ofbucillamine and allopurinol has a potent synergistic effect. Withoutbeing bound to any particular theory, one possible mechanism may be thestimulation of MRP4 (ABCC4) mediated urate transport by bucillamine andallopurinol.^(30,31,33)

It was found that bucillamine was effective in preventing neutrophiltrafficking to the peritoneum following the injection of monosodiumurate crystals. The drug also attenuated the release of IL-1β and IL-6into the peritoneal cavity. Bucillamine has been shown previously toreduce neutrophil-endothelial cell interactions in warmischaemia-reperfusion injury in the liver.⁵⁸ The most likely mechanismof action may be through the inhibition of the cytokine-inducedneutrophil chemoattractant-1 (CINC-1) which is induced by IL-1β andTNF-α and promotes both neutrophil rolling and adhesion through theupregulation of surface integrins.⁵⁸

The inventor also found, that bucillamine and colchicine synergised onthe movement of neutrophils into the peritoneum and the release of IL-1βand IL-6. The mechanism of action of colchicine is thought to be partlydue to its direct interaction with microtubules thereby inhibiting theirmigration toward the chemotactic factors.⁵⁹ Colchicine also changes thedistribution of adhesion molecules on neutrophils and endothelial cells,limiting the inflammatory response in acute gout.⁶⁰ This mechanism maybe complementary to bucillamine which is believed to attenuateneutrophil-endothelial cell interactions indirectly through theinhibition of CINC-1.⁵⁸

In preliminary experiments, it was found that bucillamine inhibited therelease of IL-1β and IL-6 from mouse macrophages in response to MSUcrystals. It has been shown previously that bucillamine attenuated therelease of TNF-α, IL-1β and IL-8 from THP-1 cells stimulated withlipopolysaccharide.⁶¹ This suggests that bucillamine may be an inhibitorof MSU crystal-induced inflammasome activation. It has been demonstratedthat the processing of IL-1β is the central event in the inflammatorycascade initiated by MSU crystals.⁵⁰

Bucillamine had an additive effect with colchicine on the release ofIL-1β from mouse macrphages following exposure to monosodium uratecrystals. Colchicine has been shown to block crystal-induced IL-1βgeneration at the level of crystal endocytosis and presentation to theinflammasome.⁵⁰

Colchicine has the narrowest therapeutic window of any acute gouttherapy and there is a considerable variability in tolerance betweenpatients.^(62, 63) However, it has been shown recently that lower dosesretain efficacy with reduced toxicity in the treatment of acute goutattacks.⁶² Colchicine has been the mainstay of treatment for goutprolphylaxis. The use of a combination of bucillamine with colchicinecould further improve the therapeutic profile of colchicine as aprophylaxis treatment especially when given chronically in order toprevent gout attacks.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

For use in therapy a therapeutically effective amount of the bucillamineand allopurinol or colchicine as defined herein, or pharmaceuticallyacceptable salts or solvates thereof, may be presented as apharmaceutical composition. Thus, in a further embodiment the inventionprovides a pharmaceutical composition according to the first, and secondaspects, in admixture with one or more pharmaceutically acceptablecarriers, diluents, or excipients. The carrier(s), diluent(s) orexcipient(s) must be acceptable in the sense of being compatible withthe other ingredients of the formulation and not deleterious to therecipient thereof.

When applicable, the compositions of the present invention, includingbucillamine and allopurinol or colchicine may be in the form of and/ormay be administered as a pharmaceutically acceptable salt.

Typically, a pharmaceutically acceptable salt may be readily prepared byusing a desired acid or base as appropriate. The salt may precipitatefrom solution and be collected by filtration or may be recovered byevaporation of the solvent.

Suitable addition salts are formed from acids which form non-toxic saltsand examples are hydrochloride, hydrobromide, hydroiodide, sulphate,nitrate, phosphate, hydrogen phosphate, dihydrogen phosphate acetate,maleate, malate, fumarate, lactate, tartrate, citrate, formate,gluconate, succinate, pyruvate, oxalate, oxaloacetate, trifluoroacetate,saccharinate, benzoate, methanesulphonate, ethanesulphonate,benzenesulphonate, p-toluenesulphonate and isethionate.

Suitable salts may also be formed from bases, forming salts includingammonium salts, alkali metal salts such as those of sodium andpotassium, alkaline earth metal salts such as those of calcium andmagnesium.

Pharmaceutically acceptable salts may also be prepared from other salts,including other pharmaceutically acceptable salts, using conventionalmethods.

Those skilled in the art of organic or coordination chemistry willappreciate that many organic and coordination compounds can formcomplexes with solvents in which they are reacted or from which they areprecipitated or crystallized. These complexes are known as “solvates”.For example, a complex with water is known as a “hydrate”.

Solvates of bucillamine, allopurinol and/or colchicine are within thescope of the present invention.

Pharmaceutical compositions of the invention may be formulated foradministration by any appropriate route, for example by the oral(including buccal or sublingual). Therefore, the pharmaceuticalcompositions of the invention may be formulated, for example, astablets, capsules, powders, granules, lozenges, creams or liquidpreparations, such as oral solutions or suspensions. Such pharmaceuticalformulations may be prepared by any method known in the art of pharmacy,for example by bringing into association the active ingredient with thecarrier(s) or excipient(s).

Tablets and capsules for oral administration may be in unit dosepresentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatine, sorbitol,tragacanth, or polyvinylpyrrolidone; fillers, for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricants, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants, for example potato starch; or acceptable wettingagents such as sodium lauryl sulphate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives, such as suspending agents, for example sorbitol,methyl cellulose, glucose syrup, gelatine, hydroxyethyl cellulose,carboxymethyl cellulose, aluminium stearate gel or hydrogenated ediblefats, emulsifying agents, for example lecithin, sorbitan, monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, oily esters such as glycerine, propylene glycol, orethyl alcohol; preservatives, for example methyl or propylp-hydroxybenzoate or sorbic acid, and, if desired, conventionalflavouring or colouring agents.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations may include other agents conventionalin the art having regard to the type of formulation in question.

The compositions of the present invention may be suitable for thetreatment of diseases in a human or animal patient. In one embodiment,the patient is a mammal including a human, horse, dog, cat, sheep, cow,or primate. In one embodiment the patient is a human. In a furtherembodiment, the patient is not a human.

As used herein, the term “effective amount” means that amount of a drugor pharmaceutical agent that will elicit the biological or medicalresponse of a tissue, system, animal or human that is being sought, forinstance, by a researcher or clinician. Furthermore, the term“therapeutically effective amount” means any amount which, as comparedto a corresponding subject who has not received such amount, results inimproved treatment, healing, prevention, or amelioration of a disease,disorder, or side effect, or a decrease in the rate of advancement of adisease or disorder. The term also includes within its scope amountseffective to enhance normal physiological function.

As used herein the term “treatment” refers to defending against orinhibiting a symptom, treating a symptom, delaying the appearance of asymptom, reducing the severity of the development of a symptom, and/orreducing the number or type of symptoms suffered by an individual, ascompared to not administering a pharmaceutical composition of theinvention. The term treatment encompasses the use in a palliativesetting

According to one embodiment of the invention, a pharmaceuticalcomposition according to the first or second aspect is used in thetreatment of gout. In one embodiment the gout is moderate to severegout. In another embodiment the gout is chronic gout. In yet anotherembodiment the gout is acute gout.

According to another embodiment of the first or second aspects of theinvention, the pharmaceutical composition comprises between 25 mg, and400 mg of bucillamine or a pharmaceutically acceptable salt or solvatethereof. Preferably, the said composition comprises between 50 mg, and300 mg of bucillamine or a pharmaceutically acceptable salt or solvatethereof. Most preferably, the said composition comprises between 50 mg,and 200 mg of bucillamine or a pharmaceutically acceptable salt orsolvate thereof.

In another embodiment, according to the first aspect of the invention,the pharmaceutical composition comprises between 200 mg and 800 mg ofallopurinol or a pharmaceutically acceptable salt or solvate thereof.Preferably, the said composition comprises between 300 mg and 600 mg ofallopurinol or a pharmaceutically acceptable salt or solvate thereof.Most preferably, the said composition comprises between 300 mg and 400mg of allopurinol or a pharmaceutically acceptable salt or solvatethereof.

In yet another embodiment, according to the second aspect of theinvention, the pharmaceutical composition comprises between 0.2 mg and1.8 mg of colchicine or a pharmaceutically acceptable salt or solvatethereof. Preferably, the said composition comprises between 0.2 mg and1.6 mg of colchicine or a pharmaceutically acceptable salt or solvatethereof. Most preferably, the said composition comprises between 0.3 mgand 1.2 mg of colchicine or a pharmaceutically acceptable salt orsolvate thereof.

In a preferred embodiment of the invention, according to the third orfourth aspects, the bucillamine and allopurinol or colchicine, orpharmaceutically acceptable salts or solvates thereof are administeredconcurrently as a single dose once a day. In another preferredembodiment, the bucillamine and allopurinol or colchicine, orpharmaceutically acceptable salts or solvates thereof are administeredorally.

According to another embodiment of the third or fourth aspects of theinvention, the said method comprises administering between 25 mg, and400 mg of bucillamine or a pharmaceutically acceptable salt or solvatethereof. Preferably, the said method comprises administering between 50mg and 300 mg of bucillamine or a pharmaceutically acceptable salt orsolvate thereof. Most preferably, the said method comprisesadministering between 50 mg, and 200 mg of bucillamine or apharmaceutically acceptable salt or solvate thereof.

In another embodiment according to the third aspect of the invention,the said method comprises administering between 200 mg and 800 mg ofallopurinol or a pharmaceutically acceptable salt or solvate thereof.Preferably, the said method comprises administering between 300 mg and600 mg of allopurinol or a pharmaceutically acceptable salt or solvatethereof. Most preferably, the said method comprises administeringbetween 300 mg and 400 mg of allopurinol or a pharmaceuticallyacceptable salt or solvate thereof.

In yet another embodiment, according to the fourth aspect of theinvention, the said method comprises administering between 0.2 mg and1.8 mg of colchicine or a pharmaceutically acceptable salt or solvatethereof. Preferably, the said method comprises administering between 0.2mg and 1.6 mg of colchicine or a pharmaceutically acceptable salt orsolvate thereof. Most preferably, the said method comprisesadministering between 0.3 mg and 1.2 mg of colchicine or apharmaceutically acceptable salt or solvate thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effects of bucillamine and allopurinol administrationon serum urate levels in hyperuricaemic mice.

FIG. 2 shows the effects of bucillamine and allopurinol administrationon the urinary excretion of uric acid.

FIG. 3 shows the effects of bucillamine and allopurinol administrationon urinary creatinine levels.

FIG. 4 shows the effect of the administration of bucillamine onmonosodium urate-induced peritoneal neutrophil influx in the mouse.

FIG. 5 shows the effects of bucillamine and colchicine administration onMSU crystal-induced peritoneal neutrophil influx in the mouse.

FIG. 6 shows the effect of bucillamine on MSU crystal-induced IL-1β andIL-6 release from mouse macrophages.

FIG. 7 shows the effect of bucillamine and colchicine in inhibitingmonosodium urate crystal-induced IL-1β release from mouse macrophages

MODES FOR CARRYING OUT THE INVENTION

In order to better understand the nature of the invention a number ofexamples will now be described as follows:

Methods

Hyperuricaemic Mice and Drug Administration

Male C57/BL/6 mice were maintained on a 12-h light/dark cycle in atemperature- and humidity-controlled room for 1 week prior to theexperiments. A total of 45 C57/Black 6 mice in 9 groups (n=5) werestudied for a period of one week. The drugs were dissolved or dispersedin distilled water immediately prior to dosing and administered in avolume of 15 mL/kg by gastric gavage. The volume of drug administeredwas based on the body weight determined on the day of dosing. In orderto produce hyperuricaema, the mice received 250 mg/kg of potassiumoxonate or water (vehicle) at 8.00 a.m. daily for seven consecutivedays.⁵¹⁻⁵³ Treatment with bucillamine, allopurinol or vehicle wasinitiated one hour after the administration of potassium oxonate andcontinued for 7 days.

Blood and Urine Collection

Whole blood and urine samples were collected 1 h after final drugadministration on the seventh day. The blood was allowed to clot forapproximately 1 h at room temperature and then centrifuged at 10,000×gfor 5 min to obtain the serum. The serum and urine were stored at −80°C. until assayed.

Determination of Uric Acid Levels

The uric acid levels in serum and urine were determined by thephosphotungstic acid method.⁵⁴

Determination of Serum Creatinine Levels

The creatinine levels in serum were determined spectrophotometricallyusing standard diagnostic kits.

Western Blot Analysis of ABCG2 in Mouse Kidney Protein

The protein samples were prepared from mouse kidney tissues using anextraction buffer containing 50 mM Tris-HCl, pH 7.4, 0.5% SDS andprotease inhibitor cocktail P8340 (Sigma), and Western blots wereperformed. The proteins (40 μg) from each sample were separated on a 10%SDS polyacrylamide gel (SDS-PAGE) and resolved at 180 volts for 1 h. Theproteins were transferred to a strip of nitrocellulose membrane. and theABCG2 protein was immunodetected by a 1:200 dilution of a primary mousemonoclonal anti-human ABCG2 antibody (Santa Cruz Biotechnology) or a1:1000 dilution of a primary mouse monoclonal anti-human BCRP (BXP-21)antibody followed by a 1:3000 dilution of a secondary donkey anti-mouseIgG-HRP antibody (Santa Cruz Biotechnology). Then ECL Plus reagents(Amersham Biosciences) were applied to the blots which were then exposedto autoradiography films (Kodak) for 3 min.

Monosodium Urate Crystal-Induced Inflammation in Mice

Monosodium urate (MSU) crystals were prepared by dissolving 1.68 g ofuric acid in 500 mL of 0.01 M NaOH and heating to 70° C.^(55,56) NaOHwas added as required to maintain the pH between 7.1 and 7.2 and thesolution was filtered and incubated at room temperature with slowstirring for 24 hours.

Male C57/BL/6 mice were treated intraperitoneally with 0.5 mg MSUcrystals in 0.5 mL of sterile PBS or PBS alone as a control.⁵⁰Bucillamine was dissolved in PBS and administered as a 50 μL i.p.injection to three groups of mice at a dose of 5, 10 or 25 μmol/kgimmediately following MSU crystal injection. Colchicine was dissolved inPBS and administered at a dose of 0.05, 0.5 and 5 μmol/kg in a similarfashion.

Colchicine was administered immediately following bucillamine in studieson the synergistic effects of the two drugs. The mice were euthanisedafter 6 hours with CO₂ and the peritoneal cells removed by lavage with10 mL of cold PBS. The lavage fluid was analysed for neutrophilinfiltration using a cytospin and Diff-Quick staining. Theconcentrations of IL-1β and IL-6 in the lavage supernatants wasdetermined using an R&D Systems Quantikine mouse immunoassay.

Mouse Peritoneal Macrophages

Male C57/BL/6 mice were injected intraperitoneally with 4%thioglycollate solution and the macrophages collected by peritoneallavage 3 days later.⁵⁷ The cells were plated at a density of 7×10⁵ cellsin 12-well dishes and non-adherent cells were removed after 3 hours. Thecells were cultured in RPMI containing 10% FCS, sodium pyruvate,penicillin/streptomycin and L-glutamine. The culture medium was replacedwith OptiMEM and the macrophages stimulated with 50 μg/mL of MSUcrystalsfor 6 hours in the presence or absence of bucillamine. The concentrationof IL-1β in the supernatant was determined with an R&D systemsQuantikine mouse immunoassay.

Results

The Effects of Bucillamine and Allopurinol on Serum and Urinary Levelsof Uric Acid and Urinary Levels of Creatinine in Hyperuricaemic andNormal Mice

The effects of bucillamine and allopurinol administration on serum uratelevels in hyperuricaemic mice are shown in FIG. 1. Bucillamine had ahighly significant (p<0.001) dose-response effect which reduced meanserum urate by 0.0067 mg/dL for each increase of 1 mg/kg/day. Theadministration of allopurinol (5 mg/kg/day)) produced a highlysignificant (p<0.001) drop in serum urate, to a level significantlybelow normal baseline (p<0.001). There was a significant (p=0.012)interactive effect between bucillamine and allopurinol. The addition ofallopurinol (5 mg/kg/day)) increased the dose-response effect ofbucillamine so that each increase of 1 mg/kg/day of bucillamine resultedin a decrease of 0.0010 mg/dL in the serum urate concentration.

FIG. 2 shows the effects of bucillamine and allopurinol administrationon the urinary excretion of uric acid. Bucillamine had a highlysignificant (p<0.001) dose-response effect which increased mean urinaryuric acid by 0.079 mg/dL for each increase of 1 mg/kg/day of the drug.Allopurinol (5 mg/kg/day) produced a highly significant (p<0.001)increase in urinary uric acid. to a level still significantly belownormal baseline (p<0.001). There was a highly significant (p<0.001)interactive effect between allopurinol and bucillamine. The addition ofallopurinol (5 mg/kg/day) increased the dose-response effect ofbucillamine such that each increase of 1 mg/kg/day of bucillamineresulted in an increase of 0.171 mg/dL in the urinary uric acidconcentration.

The results in FIG. 3 show the effects of bucillamine and allopurinoladministration on urinary creatinine levels. Bucillamine had a highlysignificant (p<0.001) dose-response effect which increased mean urinarycreatinine by 0.082 mg/dL for each increase of 1 mg/kg/day. There was ahighly significant (p=0.004) interactive effect between allopurinol andbucillamine. The addition of allopurinol (5 mg/kg/day) increased thedose-response effect of bucillamine such that each increase of 1mg/kg/day of bucillamine resulted in an increase of 0.128 mg/dL in theserum urate concentration.

The Effect of the Administration of Bucillamine on the Induction ofABCG2 in the Mouse Kidney

The Western blot analysis demonstrated that ABCG2 protein wasunregulated in the mouse kidney following treatment with a 100 mg/kg/dayof bucillamine for 7 days (data not shown). The upregulation of ABCG2 inthe presence of bucillamine may be due to the activation of Nrf2 in thekidney.^(30,31,33)

The Effect of the Administration of Bucillamine on MonosodiumUrate-Induced Peritoneal Inflammation in the Mouse

The effect of the administration of bucillamine on monosodiumurate-induced peritoneal neutrophil influx in the mouse is shown in FIG.4. Bucillamine had a highly significant (p<0.001) dose-response effectwhich decreased mean neutrophil influx by 5.15% for every increase of 1μmol/kg of the drug. A logarithmic model, leading to percentage decreaseestimates, was chosen after examining the residual plot from a standardlinear regression, which clearly displayed both curvature andheteroskedasticity. These issues were both solved by the logarithmictransformation applied to the neutrophil values.

The Effects of the Administration of Bucillamine and Colchicine onMonosodium Urate-Induced Peritoneal Inflammation in the Mouse

FIG. 5 shows the effects of bucillamine and colchicine administration onMSU crystal-induced peritoneal neutrophil influx in the mouse.Colchicine had a highly significant (p<0.001) dose-response effect whichdecreased average neutrophil influx by 18.3% for every increase of 1μmol/kg. A logarithmic model was chosen after examining the shape of therelationship between neutrophil influx and colchicine dose. It was foundthat the addition of bucillamine (10 μmol/kg) produced a highlysignificant (p<0.001) decrease in average neutrophil influx. Inaddition, there was an interactive relationship between bucillamine andcolchicine such that the addition of bucillamine enhanced thedose-response effect so that there was a decrease of 32.2% for everyincrease of 1 μmol/kg of colchicine.

The Effects of Bucillamine on Monosodium Urate-Induced IL-1β and IL-6Release from Mouse Macrophages

The effect of bucillamine on MSU crystal-induced IL-1β and IL-6 releasefrom mouse macrophages is shown in FIG. 6. Bucillamine has a highlysignificant (p<0.001) dose response effect on IL-1β cytokine production,with each additional 1 μmol/kg increase in bucillamine resulting in adecrease in average IL-1β cytokine production of 30.7 pg/mL. Bucillaminehas a highly significant (p<0.001) dose response effect on IL-6 cytokineproduction, with each additional 1 μmol/kg increase in bucillamineresulting in a decrease in average IL-6 cytokine production of 13.7pg/mL. However, this dose-response effect was significantly (p<0.001)less than the dose response effect on average IL-1β cytokine production.

The Effects of Bucillamine and Colchicine on Monosodium Urate-InducedIL-1β Release from Mouse Macrophages

The effect of bucillamine and colchicine in inhibiting MSUcrystal-induced IL-1β release from mouse macrophages is shown in FIG. 6.Colchicine had a highly significant (p<0.001) dose-response effect whichdecreased average MSU crystal-induced IL-1β production by 29.6% forevery increase of 1 μmol/kg. A logarithmic model was chosen afterexamining the shape of the relationship between IL-1β production and thedose of colchicine. The addition of bucillamine (10 μmol/kg) produced ahighly significant (p<0.001) decrease in average MSU crystal-inducedIL-1β production of 65.0%. However, there was no significant interactiverelationship between the dose of bucillamine and colchicine.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the scope of theinvention as broadly described. The present embodiments are, therefore,to be considered in all respects as illustrative and not restrictive.

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The invention claimed is:
 1. A pharmaceutical composition comprisingbucillamine or a pharmaceutically acceptable salt or solvate thereof andallopurinol or a pharmaceutically acceptable salt or solvate thereof,together with one or more pharmaceutically acceptable carriers, diluentsand/or excipients, wherein the composition is suitable for use in thetreatment of gout.
 2. The pharmaceutical composition according to claim1, wherein the said composition comprises between 25 mg and 400 mg ofbucillamine or a pharmaceutically acceptable salt or solvate thereof. 3.The pharmaceutical composition according to claim 1, wherein the saidcomposition comprises between 50 mg and 300 mg of bucillamine or apharmaceutically acceptable salt or solvate thereof.
 4. Thepharmaceutical composition according to claim 1, wherein the saidcomposition comprises between 50 mg and 200 mg of bucillamine or apharmaceutically acceptable salt or solvate thereof.
 5. Thepharmaceutical composition according to claim 1, wherein the saidcomposition comprises between 200 mg and 800 mg of allopurinol or apharmaceutically acceptable salt or solvate thereof.
 6. Thepharmaceutical composition according to claim 1, wherein the saidcomposition comprises between 300 mg and 600 mg of allopurinol or apharmaceutically acceptable salt or solvate thereof.
 7. Thepharmaceutical composition according to claim 1, wherein the saidcomposition comprises between 300 mg and 400 mg of allopurinol or apharmaceutically acceptable salt or solvate thereof.
 8. A pharmaceuticalcomposition comprising bucillamine or a pharmaceutically acceptable saltor solvate thereof and colchicine or a pharmaceutically acceptable saltor solvate thereof, together with one or more pharmaceuticallyacceptable carriers, diluents and/or excipients, wherein the compositionis suitable for use in the treatment of gout.
 9. The pharmaceuticalcomposition according to claim 8, wherein the said composition comprisesbetween 0.2 mg and 1.8 mg of colchicine or a pharmaceutically acceptablesalt or solvate thereof.
 10. The pharmaceutical composition according toclaim 8, wherein the said composition comprises between 0.2 mg and 1.6mg of colchicine or a pharmaceutically acceptable salt or solvatethereof.
 11. The pharmaceutical composition according to claim 8,wherein the said composition comprises between 0.3 mg and 1.2 mg ofcolchicine or a pharmaceutically acceptable salt or solvate thereof. 12.A method for the treatment of gout in a mammal, comprising administeringa therapeutically effective amount of bucillamine or a pharmaceuticallyacceptable salt or solvate thereof to a mammal in need thereof.
 13. Themethod according to claim 12, wherein the said method comprisesadministering between 25 mg and 400 mg of bucillamine or apharmaceutically acceptable salt or solvate thereof.
 14. The method asclaimed in claim 12, further comprising administering a therapeuticallyeffective amount of allopurinol or a pharmaceutically acceptable salt orsolvate thereof to a mammal in need thereof.
 15. The method according toclaim 14, wherein the said method comprises administering between 200 mgand 800 mg of allopurinol or a pharmaceutically acceptable salt orsolvate thereof.
 16. The method according to claim 14, wherein thebucillamine or a pharmaceutically acceptable salt or solvate thereof andallopurinol or a pharmaceutically acceptable salt or solvate thereof areadministered concurrently as a single dose once a day.
 17. The method asclaimed in claim 12, further comprising administering a therapeuticallyeffective amount of colchicine or a pharmaceutically acceptable salt orsolvate thereof, to a mammal in need thereof.
 18. The method accordingto claim 17, wherein the said method comprises administering between 0.2mg and 1.8 mg of colchicine or a pharmaceutically acceptable salt orsolvate thereof.
 19. The method according to claim 12, wherein said goutis an acute gout flare.
 20. The pharmaceutical composition according toclaim 8, wherein the composition comprises between 25 mg and 400 mg ofbucillamine or a pharmaceutically acceptable salt or solvate thereof.21. The pharmaceutical composition according to claim 8, wherein thecomposition comprises between 50 mg and 300 mg of bucillamine or apharmaceutically acceptable salt or solvate thereof.
 22. Thepharmaceutical composition according to claim 8, wherein the compositioncomprises between 50 mg and 200 mg of bucillamine or a pharmaceuticallyacceptable salt or solvate thereof.